The human carcinogenicity of almost all pesticides currently on the market is poorly evaluated and inadequately understood in spite of over 50 years of epidemiologic and toxicologic research. Most currently used pesticides are not overtly genotoxic or mutagenic in vitro and animal studies, however human studies have found suggestive evidence of increased risk for various cancers from exposure to specific pesticides. The objective of our study is to investigate whether pesticides act via an epigenetic mechanism, specifically by effecting aberrant DNA methylation, to confer increased cancer risk in humans. We hypothesize that high lifetime pesticide exposures will be associated with altered DNA methylation patterns, including global genomic hypomethylation and gene-specific hypermethylation, in a group of healthy pesticide applicators. We propose a study nested in a large, prospective cohort of licensed pesticide applicators, The Agricultural Health Study, which provides the unique opportunity afforded in few other studies to evaluate biologic responses to well characterized pesticide exposures in humans. There are no previous studies of DNA methylation measured in humans with respect to varying degrees of pesticide exposure. We will utilize blood samples from approximately 700 applicators, will extract genomic DNA, and will quantify both global DNA methylation and promoter region methylation for four of the most commonly silenced genes in cancers of concern to this cohort, p16INK4, MGMT, CDH-1, and GSTp1. Our primary aim is to investigate whether exposure to specific pesticides and chemical classes of pesticides predicts (1) hypermethylation of specific genes (p16INK4, MGMT, CDH-1, and GSTp1) and (2) decreased global genomic methylation (hypomethylation) estimated in Alu family of CpG repeats and LINE-1 (long interspersed nucleotide element-1) sequences of DNA in blood cells. Our secondary aims are to explore whether other farm related exposures, such as solvents, grain dust, welding fumes, and high pesticide exposure events modify the associations between pesticide exposures and gene specific or global methylation and to explore and elucidate patterns of gene specific and global methylation according to baseline characteristics, such as age, recent and past tobacco smoking, alcohol consumption and diet among a low pesticide exposure group. Our proposed study has the potential to identify a previously unexplored, potential mechanism of carcinogenicity through which pesticides may operate in humans. PUBLIC HEALTH RELEVANCE: The extent of worldwide pesticide usage and underscores the potential for these exposures to have enormous public health implications. Many pesticides currently on the market and in widespread use by the American public pass safety testing based on genotoxicity and mutagenicity assays, however epidemiologic studies suggest carcinogenicity in humans. Our proposed study will address a significant gap in our knowledge of pesticide carcinogenicity, specifically whether pesticides operate via an epigenetic mechanism, such as DNA methylation to confer increased cancer risk in humans.